The realization that HIV-1 eradication is impossible with current treatment raises two fundamental questions about HIV pathogenesis: how and where does the virus sustain itself as a life-long persistent infection? Our preliminary data suggest lymphoid tissues (LT), i.e., gut associated lymphoid tissue (GALT) and secondary lymph nodes (LN), are a major site of HIV persistence. We found that CD4+ cells are rapidly depleted in GALT and LN soon after HIV acquisition and there is poor CD4 reconstitution with antiretroviral therapy (ARV). In these patients with HIV RNA in plasma < 400 or 50 copies.ml, we have also found: 2 LTR episomes (a marker for recent HIV replication) in GALT and LN, frequencies of HIV DNA+ cells in GALT increase, drug penetration into LN and GALT is impaired, and depletion of central memory CD4 cells in GALT may correlate to frequency of reactivation of chronic latent mucosal pathogens like herpes simplex 2 (HSV2). These data suggest this cryptic HIV replication in LT is a potential reservoir of importance and provide the basis for 3 inter-related hypotheses: 1) cryptic replication occurs in LT and fuels viral recrudescence with treatment interruption, 2) it is a consequence of inadequate concentration of drug in CD4 cells of GALT and LN, and 3) it eventually undermines immune function in GALT and LN. The critical predictions and thus key measurements to test these hypotheses and their corollaries are to determine the extent of cryptic HIV replication in GALT and LN in relation to concentrations of active drug levels and assess the impact of ongoing replication on immune function in GALT and LN. Dr. Stevenson will isolate viral and episomal DNA from all compartments to establish the existence of a LT reservoir and perform sequence analysis to determine relatedness of virus appearing in PB after treatment interruption to the cryptic reservoir in LT. Dr. Haase will use in situ hybridization, laser capture microdissection, and in situ per to establish the size and location of persistent reservoirs of infection in each compartment. Dr. Fletcher will determine ARV levels in GALT and LN CD4+ cells, look for evidence of efflux mechanisms causing decreased intracellular concentration of drug, and estimate the concentration of drug in LT required to fully suppress cryptic replication. Dr. Schacker will determine the absolute size of CD4 subpopulations in each compartment and relationships between levels of cryptic replication, CD4 depletion in GALT, and measures of mucosal reactivation of mucosal pathogens. Dr. Douek will perform flow cytometry to measure antigen specific responses and cell separation and culture virus from CD4 cells obtained from each compartment before and during treatment interruption. If our hypotheses are correct it will point the way toward improved therapies whose impact will be measured by improved control of viral replication and preservation of immune function in the relevant LT reservoirs.